Figure 1. U-DNA-Seq provides genome-wide mapping of uracil-DNA distribution.

(A) Schematic image of the novel U-DNA immunoprecipitation and sequencing method (U-DNA-Seq). After sonication, enrichment of the fragmented U-DNA was carried out by the 1xFLAG-ΔUNG sensor construct followed by pull-down with anti-FLAG agarose beads. U-DNA enrichment compared to input DNA was confirmed by dot blot assay before samples were subjected to NGS. (B) Immunoprecipitation led to elevated uracil levels in enriched U-DNA samples compared to input DNA in case of both 5FdUR (5FdUR_UGI) and RTX (RTX_UGI) treated, UGI-expressing HCT116 samples. For each treatment, the same amount of DNA was loaded as input and enriched U-DNA samples providing a correct visual comparison. Two-third serial dilutions were applied.

Figure 1—figure supplement 1. Elevation of genomic uracil content upon stable UGI expression and drug treatments.

(A) HCT116 cells stably expressing UGI along with EGFP following retroviral transduction. GFP positive cells were selected by fluorescence-activated cell sorting and cultured for further analysis. DAPI was used for DNA staining. Scale bar represents 20 µm. (B) Dot blot assay for measuring genomic uracil levels (Róna et al., 2016) of non-treated and drug (5FdUR or RTX) treated HCT116 cells either transiently or stably expressing UGI. Genomic DNA (8 ng) isolated from log-phase growing CJ236 [dut-, ung-] Escherichia coli strain was applied as uracil standard in a ½ dilution series (upper panel). Two-third dilution series from HCT116 samples started with 600 ng DNA for non-treated or 5 ng DNA for 5FdUR or RTX treated samples (lower panel). The dot blot image presented is a representative of six independent biological experiments. (C) Bar graph shows the uracil moieties/million bases of each sample. Drug treatment led to significantly elevated uracil levels in HCT116 cells either transiently or stably expressing UGI (~400 uracil moieties/million for 5FdUR and ~700 uracil moieties/million for RTX) as compared to non-treated (NT) cells (~2–5 uracil moieties/million). Error bars indicate standard errors of the mean (SEM). Calculations were based on six independent datasets (n = 6). p=0.002. Source data are available in Figure 1—figure supplement 1—source data 1. (D) Bar graph shows the uracil moieties/million bases in MMR proficient HCT116 cells stably expressing UGI. Drug treatment resulted in even higher U-DNA content (~900 uracil moieties/million for 5FdUR and ~950 uracil moieties/million for RTX) as compared to MMR deficient cells. Error bars indicate standard errors of the mean (SEM). Calculations were based on six independent datasets (n = 6). p=0.005. Source data are available in Figure 1—figure supplement 1—source data 1.

Figure 1—figure supplement 2. Negative control of U-DNA-IP using ΔUNG sensor free (empty) anti-FLAG beads.

(A) Relative amount of the pulled down DNA as compared to the input DNA amount. Samples were the following: U-DNA-IP from UGI-expressing, non-treated (NT_UGI) and 5FdUR treated (5FdUR_UGI) samples, and the corresponding mock pulled down controls (NT_UGI_ctr and 5FdUR_UGI_ctr) where ΔUNG sensor free, empty anti-FLAG beads were used. (B) Pearson correlation among log2 ratio coverage tracks calculated from the genome scaled input coverage track and either the original (NT_UGI_no_ctr and 5FdUR_UGI_no_ctr) or the control-subtracted (NT_UGI_ctr_subtracted and 5FdUR_UGI_ctr_subtracted), enriched coverage tracks (Materials and methods, Supplementary file 1). (C) A representative IGV view on original (NT_UGI_no_ctr and 5FdUR_UGI_no_ctr) and the control-subtracted (NT_UGI_ctr_subtracted and 5FdUR_UGI_ctr_subtracted) log2 enrichment (on chr3: 26,000,000–62,000,000).